Expanded metabolic versatility of ubiquitous nitrite-oxidizing bacteria from the genus Nitrospira Hanna Kocha, Sebastian Lückera,1, Mads Albertsenb, Katharina Kitzingera, Craig Herbolda, Eva Spieckc, Per Halkjaer Nielsenb, Michael Wagnera, and Holger Daimsa,2 aDepartment of Microbiology and Ecosystem Science, Division of Microbial Ecology, Research Network Chemistry meets Microbiology, University of Vienna, 1090 Vienna, Austria; bCenter for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, 9220 Aalborg, Denmark; and cBiozentrum Klein Flottbek, Microbiology and Biotechnology, University of Hamburg, 22609 Hamburg, Germany Edited by James M. Tiedje, Michigan State University, East Lansing, MI, and approved July 29, 2015 (received for review April 2, 2015) Nitrospira are a diverse group of nitrite-oxidizing bacteria and Nitrobacter genome sequences provided first insights into the among the environmentally most widespread nitrifiers. How- genomic makeup of NOB and revealed a greater metabolic ever, they remain scarcely studied and mostly uncultured. Based flexibility than anticipated earlier, which included the mixotrophic on genomic and experimental data from Nitrospira moscoviensis utilization of various organic substrates (8, 9). However, Nitro- representing the ubiquitous Nitrospira lineage II, we identified bacter require high nitrite concentrations (10, 11), and molecular ecophysiological traits that contribute to the ecological success surveys indicated that Nitrobacter are not the primary NOB in Nitrospira N. moscoviensis of . Unexpectedly, possesses genes coding ecosystems with low ambient nitrite levels such as unfertilized soils for a urease and cleaves urea to ammonia and CO2.Ureolysiswasnot (12), freshwater habitats (13), and most WWTPs (14). N. moscoviensis observed yet in nitrite oxidizers and enables to sup- Among all known NOB, the genus Nitrospira appears to be ply ammonia oxidizers lacking urease with ammonia from urea, most widespread in different habitat types and is phylogeneti- which is fully nitrified by this consortium through reciprocal feeding. cally most diverse. Nitrospira are well adapted to low nitrite The presence of highly similar urease genes in Nitrospira lenta from concentrations (10, 11) and form at least six phylogenetic line- activated sludge, in metagenomes from soils and freshwater ages (15, 16) that are globally distributed in soils (17, 18), the habitats, and of other ureases in marine nitrite oxidizers, sug- oceans (19), freshwater habitats (20), hot springs (16), and many gests a wide distribution of this extended interaction between MICROBIOLOGY ammonia and nitrite oxidizers, which enables nitrite-oxidizing other oxic habitats (15). In addition, Nitrospira members are the bacteria to indirectly use urea as a source of energy. A soluble key NOB in most WWTPs (14, 15). Nitrospira are notoriously formate dehydrogenase lends additional ecophysiological flexi- difficult to culture under laboratory conditions and, hence, de- bility and allows N. moscoviensis to use formate, with or without spite their ecological and biotechnological importance, little is concomitant nitrite oxidation, using oxygen, nitrate, or both com- known about their ecophysiology. Interestingly, not all members of pounds as terminal electron acceptors. Compared with Nitrospira this genus are restricted to nitrite as their sole source of energy defluvii from lineage I, N. moscoviensis shares the Nitrospira core and reductant. Some Nitrospira from marine ecosystems or acti- metabolism but shows substantial genomic dissimilarity including vated sludge can use simple organic substrates, such as pyruvate, genes for adaptations to elevated oxygen concentrations. Recipro- cal feeding and metabolic versatility, including the participation in Significance different nitrogen cycling processes, likely are key factors for the Nitrospira niche partitioning, the ubiquity, and the high diversity of Nitrification, the sequential aerobic oxidation of ammonia via in natural and engineered ecosystems. nitrite to nitrate, is a key process of the biogeochemical ni- trogen cycle and catalyzed by two aerobic microbial guilds Nitrospira | nitrification | genome | urease | formate (nitrifiers): ammonia oxidizers and nitrite-oxidizing bacteria (NOB). NOB are generally considered as metabolically restricted itrification, a key aerobic process of the biogeochemical and dependent on ammonia oxidizers. Here, we report that, Nnitrogen (N) cycle, is catalyzed by two guilds of chemo- surprisingly, key NOB of many ecosystems (Nitrospira) convert lithoautotrophic microorganisms. The ammonia-oxidizing mi- urea, an important ammonia source in nature, to ammonia and croorganisms (bacteria and archaea; AOM) oxidize ammonia to CO2. Thus, Nitrospira supply urease-negative ammonia oxi- nitrite, which is subsequently oxidizedtonitratebynitrite- dizers with ammonia and receive nitrite produced by ammonia oxidizing bacteria (NOB). Nitrification links aerobic and anaero- oxidation in return, leading to a reciprocal feeding interaction bic pathways of the N cycle by providing nitrate or nitrite as of nitrifiers. Moreover, Nitrospira couple formate oxidation electron acceptors for dissimilatory nitrate reduction, denitrifica- with nitrate reduction to remain active in anoxia. Accordingly, tion, respiratory ammonification, and anaerobic ammonium Nitrospira are unexpectedly flexible and contribute to nitrogen oxidation (1, 2). The end product of nitrification, nitrate, is an cycling beyond nitrite oxidation. important source of nitrogen for assimilation by many microor- ganisms and plants. Moreover, nitrification is a key step of bi- Author contributions: H.K., S.L., P.H.N., and H.D. designed research; H.K., S.L., M.A., and K.K. performed research; E.S. contributed new reagents/analytic tools; H.K., S.L., M.A., ological wastewater treatment but also contributes to N losses K.K., C.H., and H.D. analyzed data; and H.K., M.W., and H.D. wrote the paper. from fertilized agricultural soils (3). Being a two-step process that The authors declare no conflict of interest. involves two functional groups, nitrification is a prime example of This article is a PNAS Direct Submission. a tight metabolic interaction between free-living microorganisms. Freely available online through the PNAS open access option. Current insights into the ecology of chemolithoautotrophic Data deposition: The sequences reported in this paper have been deposited in the GenBank NOB suggest that two of the six known NOB genera are re- database (accession nos. CP011801 and KR873362–KR873392). stricted to marine ecosystems (Nitrospina and Nitrococcus) (4). 1Present address: Department of Microbiology, Radboud University, 6525 AJ Nijmegen, The recently identified Nitrolancea was enriched from activated The Netherlands. sludge from a wastewater treatment plant (WWTP) (5), whereas 2To whom correspondence should be addressed. Email: [email protected]. Nitrotoga occur in soils and WWTPs (6, 7). Nitrobacter are gen- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. erally common in terrestrial and limnic habitats. Analyses of 1073/pnas.1506533112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1506533112 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 formate, and glycerol, for carbon assimilation and probably also as was cleaved by the cytoplasmic urease (Fig. S1B). Ammonia then energy sources in addition to CO2 and nitrite (mixotrophy) (15, 19, diffused through the cell membrane into the medium or am- 21, 22). Nitrospira moscoviensis even grows by aerobic hydrogen monium was exported by AmtB transporters, which are encoded oxidation as an alternative lifestyle outside the N cycle (23). by three amtB genes in the close vicinity of the urease genes (Fig. Furthermore, this organism can reduce nitrate with H2 as an S1A). Consistent with the lack of urease genes in the genome of electron donor, but under these conditions, growth was not de- N. defluvii, no ureolytic activity was observed during incubation tected (24). of this Nitrospira strain in medium containing urea (Fig. S2A). So far, only one study analyzed a fully sequenced Nitrospira genome, which was obtained from N. defluvii (25). This Nitrospira Ureolytic Activity of NOB Drives Full Nitrification. Urea is an im- lineage I member had been enriched from a WWTP (26). Here, portant dissolved organic N compound in marine (coastal and we analyzed the genome of N. moscovienis representing Nitrospira open water) and freshwater ecosystems, where it is produced by lineage II, which is the Nitrospira clade most widely distributed in heterotrophic bacteria and also released by phytoplankton, mi- both natural and engineered ecosystems (15). This newly sequenced crofauna, and macrofauna (29). Because commercially synthe- Nitrospira genome revealed surprising metabolic features that were sized urea plays a major role as plant fertilizer, urea is globally experimentally confirmed. These findings change the current per- widespread in agricultural soils. Fertilization also contributes to ception on the interdependence of nitrifiers and demonstrate an increased urea levels in coastal waters due to riverine input. In unexpected ecophysiological versatility of Nitrospira with contri- addition, a large portion of the N in municipal wastewater occurs butions to N-cycling processes other than nitrite oxidation. as urea. Despite the environmental abundance of urea, not